Publications and other reference materials referred to herein are incorporated herein by this reference. The following description of the background of the invention is intended to aid in the understanding of the invention, but is not admitted to describe or constitute prior art to the invention.
The near infrared absorption and emission of prophyrins, phthalocyanines and other azaporphyrins and certain other aromatic nitrogen containing macrecycles have for some time made these compounds attractive candidates for use as fluorescence labels.
The phthalocyanines, particularly because of their strong near infrared absorption (molar extinction coefficients about 200,000) their high quantum yields and the resistance to fading of common metallophthalocyanine dyes have given rise to many efforts to utilize them as fluorescent labels. However, earlier efforts along these lines did not yield entirely satisfactory products largely because of the unusually strong tendency of phthalocyanines to associate, particularly by stacking in face to face aggregates, and also to bind strongly to a variety of other molecular surfaces (nonspecific binding).
As a result of intramolecular stacking unsubstituted phthalocyanines have very low solubilities in both organic and aqueous solvents. As is now well known, the tendency to stack can be reduced by the introduction of charged groups, such as sulfonate. While phthalocyanines with such substituents may possess high solubility in water and in aqueous solutions of electrolytes, the tendency to bind nonspecifically largely persists. Much of the scientific interest in fluorescence labeling is focused on applications involving biological materials such as tissue sections, cells, cell fragments, proteins, including glycol- and lipo-proteins, peptides, oligo- and poly-saccharides, oligo- and poly-nucleotides and lipids. A tendency to bind nonspecifically in fluorescence assays involving these materials may interfere by partially masking the specific interactions of interest. The nonspecific binding as well as the tendency to stack can be reduced to levels negligible in assays for therapeutic drugs by coupling the phthalocyanine dye to one or more polyoxyhydyocarbyl groups, typically methoxy-terminated poly (ethylene glycol), (PEG). At the same time the attachment of such groups preserves the desirable absorption and emission characteristics. The same technology is also effective for a wide variety of other near infrared dyes. See, U.S. Pat. No. 5,403,928.
Further significant advances have been made in the ability to measure the relevant parameters in immunoassays. For example, using the technique described in Dandliker et al, U.S. Pat. No. 5,302,349, entitled “Transient State Luminescence Assay Apparatus” incorporated here by reference, in its entirety, including any drawings, allows the concentrations of the bound and free forms of the components to be determined in a homogeneous assay format, i.e. no separation of bound and free is required.
Despite the significant and promising improvements made in the field of fluorescent labels and in the data analysis aspect there remains a need in the art for additional dyes which have the essential advantages but are also easier to prepare and have greater chemical stability. Prior closely related art by others is to be found in U.S. Pat. Nos. 5,135,717, 5,346,670, and 5,494,793.